This invention relates to the Sox-9 (SOX-9 in humans) gene which appears to have a role in mammalian skeletal development and which is also related to the inherited skeletal disease syndrome Campomalic Dysplasia (CD), alternatively known as campomelic dwarfism or campomelic syndrome.
CD is an osteochondrodysplasia affecting 0.05-2.2 per 10,000 live births. It is characterised by congenital bowing and angulation of the long bones, together with other skeletal defects, The scapulae are very small and the pelvis and the spine show changes. One pair of ribs is usually missing. Severe anomalies of the lower cervical spine are seen. The interior part of the scapula is hypoplastic. Cleft palate, micrognethia, flat face and hypertension are also features. Various defects of the ear have been noted, affecting the cochlea, malleus, incus, stapes and tympanum. Most patients die in the neonatal period of respiratory distress which has been attributed to hypoplsia of tracheobronchial cartilage (Lee et al., 1972, Am. J. Dis. Child, 124, 485-496) and small thoracic cage (Houston et al., 1983, Am. J. Med. Genet., 15, 3-28).
The human SOX-9 gene has been mapped to chromosome 17 within a region which also contains CMPD1, the locus for CD.
Chromosomal localisation of CMPD1 was based on three independent, apparently balanced, de novo reciprocal translocation involving chromosome 17 (Tommerup et al., 1993, Nature Genet., 4, 170-174). All three translocations had breakpoints between 17q24 and q25, distal to the growth hormone locus (GH) but proximal to thymidine kinase (TK-1). This mapping excluded previous CMPD1 candidates HOX2 and COL1A1. Mutations within the SOX-9 gene have now been found in DNA from CD patients (Foster et al., Nature, in press; Wagner et al., Cell, in press) proving that the SOX-9 gene has a role in skeletal development. Curiously, CD is often associated with sex reversal (Hovmoller et al., 1977, Hereditas, 86, 51-62). Among 33 cases with CD and an XY karyotype, 21 were phenotypic females and two were intersexes (Houston et al., 1983, supra). This association defines an autosomal sex-reversal locus SRA1 at or near the CMPD1 locus.
Recurrent observations of CD in sibs and occasional consanguinity in CD-affected families have led to the belief that CD is inherited as an autosomal recessive disorder. However, a total of five independent de novo chromosomal rearrangements associated with CD lends some support to a dominant, usually lethal mutation (Tommerup et al., 1993, supra). This may explain a case of CD affecting a mother and daughter, although it is possible that the milder phenotype in these patients represents a different mutation (Lynch et al., 1993, J. Med. Genet., 30, 683-686).
The murine Sox-9 gene has been mapped to distal mouse chromosome 11. This region contains various disease loci including Ts, the locus for the mouse mutant Tail-short.
Tommerup et al., 1993, above, have noted the similarities between CD and Tail-short (Ts), which also maps between Gh and Tk-1 of mouse chromosome 11 (Buchberg et al., 1992, Mammal, Genome, 3, 5162-181). No sex reversal has been associated with Ts. It is not yet clear whether the same gene is affected in both CD and Tail-short. The similarity between the two phenotypes raises the intriguing possibility that the human mutation would be homozygous lethal at the blastocyst stage, with heterozygosity resulting in the campomelic phenotype.
Ts is a mouse developmental mutant first described by Morgan, 1950, J. Hered., 41, 208-215. The mutation is semi-dominant: homozygotes die at the blastocyst stage, before or shortly after implantation (Paterson, 1980, J. Expt. Zool., 211, 247-256). Heterozygotes are small with kinked tails and numerous other skeletal defects. The phenotype is variable, but typical abnormalities have been described (Deol, 1961, Proc. R. Soc. Lon. B., 155, 78-95). The short, kinked tail is caused by reduced number and dysmorphology of caudal vertebrae. Vertebral fusions and dyssymphyses also affect the presacral and sacral regions. The humerus, tibia, and to a lesser extent femur and radius are affected by shortening and in some cases bending. Anomalies of the feet are common. These include triphalangy of digit I, absence of falciform, and various digital and other fusions. Additional ribs and rib fusions, and various skull abnormalities are evident.
Despite the obvious effects on the skeletal system in Tail-short and CD, there is some debate as to the nature of the primary defect. Ts is associated with anaemia and general growth retardation appearing at day 9, two days before the first signs of skeletal abnormality appear (Deol, 1961, above). CD is associated with vascular defects and aberrant musculature (Rodiguez, 1993, Am. J. Med. Genet., 46, 185-192) and has been mimicked in avian and amphibian embryos by teratogens affecting the nervous system (Roth, 1991, Paedr. Radiol., 21, 220-225).
SOX-9 encodes one of a family of transcription factors related to the mammalian Y-linked testis determining factor Sry. The cloning of the Y-linked testis determining gene (SRY in humane, Sry in mice) in 1990 (Gubbay et al., 1990, Nature, 346, 245-250; Sinclair et al., 1990, Nature, 346, 240-244) and subsequent demonstration that its expression is sufficient to cause male development in chromosomally female (XX) mice (Koopman et al., 1991, Nature, 351, 117-121) represented a breakthrough in positional cloning and developmental biology. The protein product of Sry contains a 79 amino acid motif that had already been detected in several other proteins, notably the high mobility group (HMG) of nuclear proteins (Jantzen et al., 1990, Nature, 344, 830-836). Several known sequence-specific DNA binding proteins contain a similar motif. Recent evidence that SRY can bind directly to DNA in a sequence-specific manner (Giese et al., 1992, Science, 255, 453-456) supports the contention that Sry acts as a transcription factor.
When a probe corresponding to the HMG box region of human SRY was hybridised to Southern blots of mouse DNA, a large number of bands was seen in addition to the strongly hybridising, Y-specific band representing mouse Sry (Gubbay et al., 1990, supra). These additional bands are present in both XX female and XY male, DNA, suggesting that there are genes related to Sry by the HMG box, present on autosomes and/or the X chromosome. Indeed, screening of cDNA libraries with an HMG box probe derived from Sry yielded four classes of hybridising clone, none of them Y-linked. Sequencing of these clones showed that they are highly related to each other (78-98% amino acid homology in the HMG box region) as well as to Sry (77-82%). They are less closely related to other mammalian genes containing HMG boxes (around 50% amino acid homology in the HMG box region). These non-Y-linked homologues of Sry have been named Sox genes (Sry-type HMG box genes). Together with Sry, the Sox genes represent a distinct family of mouse genes that appear to encode transcription factors. Western blotting using an antibody to the SRYHMG box suggests that the number of SOX genes may be as high as 50.
cDNA clones corresponding to genes dubbed Sox-1 to -4 were isolated from an 8.5 days post coitum (dpc) mouse embryo library (Gubbay et al., 1990, supra), raising speculation that they play a role in developmental decisions in the mammalian embryo. These genes were expressed throughout the CNS at first, and later become restricted to subsets of nervous tissue such as the developing eye and ear. It appears that Sox-1 to -3 are involved in specifying the development of the central nervous system. Sox-4 acts as a transcriptional activator in T-lymphocytes (van de Wetering et al., 1993, EMBO J., 12, 3847-3854). Sox-5 is expressed stage-specifically in round spermatids in the adult testis, suggesting a role in spermatogenesis, and was also shown to bind DNA in vitro (Denny et al., 1992, EMBO J., 11, 3705-3712). Denny et al., 1992, Nucleic Acids Res., 20, 2887, identified two further Sox sequences. Sox-6 and Sox-7, but corresponding cDNAs have yet to be cloned and their expression has not been characterised.
A further 10 members of the mouse Sox gene family have been identified. Degenerate primers were made corresponding to highly conserved regions at the ends of the HMG box of Sry and known Sox genes. Total RNA was prepared from 11.5 days post coitum (dpc) mouse embryos and reverse transcriptase polymerase chain reaction (RT-PCR) was performed using the degenerate primers. The PCR products were cloned and sequenced to reveal seven novel genes which have been called Sox-8, -9, -10, -11, -12, -13 and -14 (Wright et al., 1993, Nucleic Acids Res., 21, 744). Three more Sox sequences have also been isolated (Sox-16, -17 and -18) from macrophage and muscle cDNA (Layfield et al., unpublished data). Sequence comparison of the mouse Sox gene family in regard to the HMG box indicates that the Sox genes fall into seven distinct sub-groups; A: Sry; B: Sox-1, -2, -3 and -14; C: Sox-4, -11 and -12; D: Sox-5, -6 and -13; E; Sox-8, -9 and -10; F: Sox-7, -17 and -18; G: Sox-15 and -16. Whether this structural sub-grouping is reflected in the functions of these genes remains to be determined, but there is every indication that Sox genes represent a major development gene family, similar in many respects to the Hox and Pax families of developmental genes.
The conclusion that Sox genes play an important role in development is reinforced by the finding that multiple Sox genes are present in the genomes of many non-mammalian species. Six Sry-related sequences have been described in the lesser black-backed gull Larus fuscus, nine in American alligator, five in lizards, eight in chickens, seven in Drosophila and three in frogs (Griffiths, 1991, Phil. Trans. Roy. Soc. Lond. B., 244, 123-128; Denny et al., 1992, Nucleic Acids Res. above, Coriat et al., 1993, PCR Meth. App., 2, 218-222). Sox genes are widespread within the class mammalia. Sox-3 was recently cloned in marsupials (Foster and Graves, 1994, Proc. Natl. Acad. Sci. USA., 91, 1927-1931), and 12 human SOX genes have been identified (Denny et al., 1992, Nucleic Acids Res., above; Farr et al., 1993, Mammal. Genome, 4, 577-584; Goze et al., 1993, Nucleic Acids Res., 21, 2943; Stevanovic et al., 1993, Human Mol. Genet., 3, 2013-2018).
Articles by Sinclair et al. (1990, Nature, 346, 240-244), Koopman et al. (1991, Nature, 351, 117-121) and Goodfellow and Lovell-Badge (1993, Ann. Rev. Genet., 27, 71-92) referred to hereinafter also confirm that SRY is a dominant inducer of testis development in mammals. Since the discovery of SRY, many other genes have been identified that encode related HMG boxes.
The identification and cloning of SRY depended on the investigation of the genomes of patients with sex reversal syndromes, some with chromosomal rearrangements. In addition to SRY on the human Y chromosome, at least five autosomal and one X-linked loci have also been linked with XY female sex reversal and the failure to develop a testis (Bernstein, R. et al., 1980, J. Med. Genet., 17, 291-300; Pelletier, J. et al., 1991, Nature, 353, 431-434; Bennett, C. P. et al., 1993, J. Med. Genet, 30, 518-520; Wilkie, A. O. M. et al., 1993, Am. J. Med. Genet, 46, 597-600; Bardoni, B. et al., 1994, Nat. Genet, 7, 497-501; Luo, X. et al., 1994, Cell, 77, 481-490). Four of these loci have been defined by the study of rare chromosomal rearrangements. Duplications of the X chromosome short arm cause XY female development (Bernstein, R. et al, 1980, supra). The sex reversal in these patients results from the presence of two active copies of DSS (dosage sensitive sex reversal gene) which maps to a 160 kb region of Xp21 (Bardoni, B. et al., 1994, supra). Autosomal loci on chromosome 9p and on 10q have been implicated by chromosomal deletions in XY females (Bennett, C. P. et al., 1993, supra; Wikie, A. O. M. et al., 1993, supra). It is not known if the sex reversal in these instances is due to monosomy for dosage sensitive genes or whether the deletions reveal recessive mutations. A third autosomal locus, SR41, is on chromosome 17 (Tommerup, N. et al., 1993, supra) and, in this case, the sex reversal is associated with CD. The diagnosis of CD is not entirely straightforward. The most conspicuous feature is congenital bowing and angulation of the long bones. However, this type of bowing is also seen in other skeletal dysplasias (McKusick, V. A., 1992, Mendelian Inheritance in Man., The Johns Hopkins Press, Baltimore). Other features may include a variety of skeletal deformities associated with bone and cartilage formation. Patients usually die in the first week of life from respiratory failure, however, the severity of the phenotype is variable and a few patients are mildly affected and survive into adult life. A striking feature of CD is the associated sex reversal. To date there have been at least 121 reported cases of CD. Of those that have been karyotyped, 24 are 46,XX females, 14 are 46,XY males. 34 are 46,XY females (with a gradation of genital defects) and two are cases of ambiguous genitalia with an XY karyotype (Tommerup, N. et al., 1993, supra; Young, I. D. et al., 1992, J. Med. Genet, 29, 251-252; Houston, C. S., et al., 1983, supra). The remaining 47 non-karyotyped cases show a skewed sex ratio of 31:16 in favour of females. Some of the sex reversed cases examined histologically exhibit gonadal dysgenesis implying that the gene(s) responsible for CD also plays a part in testis formation.
The inheritance pattern of CD is not obvious. Many reviewers have concluded that autosomal recessive inheritance is the most likely (Cremin, B. J., et al., 1973, Lancet, 1, 488-489), although it is difficult to distinguish this pattern from autosomal dominant inheritance with variable penetrance. Similarly, it is not clear if the bone malformation and sex reversal are caused by mutation of a single gene or of a pair of linked genes in a contiguous gene syndrome. Five chromosomal rearrangements associated with CD and sex reversal have been reported which localise the gene(s) responsible to the long arm of human chromosome 17 (Tommerup, N. et al., 1993, supra; Young, I. D. et al., 1992, supra; Maraia, R. et al., 1991, Clin. Genet, 39, 401-408). Recently, Tommerup et al., 1993, supra have refined this localisation to 17q24.1-25.T with GH and TK as flanking markers. A high resolution nap has been constructed across this 20 Mb region using a panel of whole genome radiation hybrids. The map has been used to position the translocation breakpoint from a 46,XY,t(2; 17)(q35;q23-24) sex reversed campomelic dysplasia individual (Patient E) (Young, I. D. et al., 1992, supra).
It has now been found that DNA sequences of the Sox-9 and SOX-9 genes have now been elucidated and thus preparation of recombinant proteins encoded by these genes can be facilitated. An isolated DNA molecule combining these sequences and/or the recombinant proteins can be utilised therapeutically in relation to regeneration of bone or cartilage as described hereinafter.
Therefore, in one aspect, the invention provides an isolated DNA molecule comprising a DNA sequence selected from a group consisting of:
(i) a sequence of nucleotides as shown in FIG. 1 (SEQ ID NO:18);
(ii) a sequence complementary to the sequence according to (i); and
(iii) a sequence having up to 21% variation from the sequences according to (i) or (ii) which sequence is capable of hybridising thereto under standard hybridisation conditions which codes for a polypeptide of the SOX-9 type.
In another aspect, the invention provides an isolated DNA molecule comprising a DNA sequence selected from a group consisting of:
(a) a sequence of nucleotides as shown in FIG. 8a (SEQ ID NO:20);
(b) a sequence complementary to the sequence according to (a); and
(c) a sequence having up to 18% variation from the sequences according to (a) or (b) which sequence is capable of hybridising thereto under standard hybridisation conditions and which code for a polypeptide of the SOX-9 type.
The Invention also provides recombinant proteins encoded by both the Sox-9 gene and the SOX-9 gene as described hereinafter.
The Sox-9 sequence (iii) discussed above and the SOX-9 sequence (c) discussed above correspond to hybrids of the DNA sequences shown in FIGS. 1 and 8a (SEQ ID NOS:18 and 20) as such hybrids may be isolated by standard hybridisation methods as described in Sambrook et al. (1989, in Molecular Cloning: A Laboratory Manual Cold Spring Harbour Laboratory Press, New York; in particular sections 9.31 to 9.59), or direct sequence comparison.
Hybrids of the above mentioned sequences may be prepared by a procedure including the steps of:
(i) designing primers which are preferably degenerate which span at least a fragment of the relevant DNA sequences referred to above; and
(ii) using such primers to amplify said at least a fragment either from an original cDNA library or cDNA reverse transcribed from either poly A+ RNA or total RNA which RNA is derived from an appropriate source referred to herein.
The recombinant protein may be prepared by a procedure including the steps of:
(a) ligating a DNA sequence encoding a recombinant protein of the SOX-9 type or biological fragment thereof into a suitable expression vector to form an expression construct;
(b) transfecting the expression construct into a suitable host cell;
(c) expressing the recombinant protein; and
(d) isolating the recombinant protein.
The vector may be a prokaryotic or a eukaryotic expression vector.
Suitably, the vector is a prokaryotic expression vector.
Preferably, the vector is pTrcHisA.
The host cell for expression of the recombinant protein can be a prokaryote or eukaryote.
Suitably, the host cell is a prokaryote.
Preferably, the prokaryote is a bacterium.
Suitably, the bacterium is Escherichia coli. 
Alternatively, the host cell may be a yeast or a baculovirus.
The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al., (1989, supra, in particular Sections 16 and 17).
In yet another aspect, the invention provides a method of regeneration of bone or cartilage by administration of a DNA molecule or protein referred to above to a subject suffering from bone or cartilage deficiency.
Preferably the DNA molecule or protein may be injected directly into joint tissue such as knees, knuckles, elbows or ligaments. Therefore, the compounds of the invention may be utilised as a therapeutic agent in regard to treatment of cartilage or bone damage caused by disease or aging or by physical stress such as occurs through injury or repetitive strain, e.g. xe2x80x9ctennis elbowxe2x80x9d and similar complaints. The therapeutic agent of the invention may also be utilised as part of a suitable drug delivery system to a particular tissue that may be targeted. Other therapeutic applications for the compounds of the invention may include the following:
1. Use in cartilage and/or bone renewal, regeneration or repair so as to ameliorate conditions of cartilage and/or bone breakage, degeneration, depletion or damage such as might be caused by aging, genetic or infectious disease, wear and tear, physical stress (for example, in athletes or manual labourers), accident or any other cause, in humans, livestock, domestic animals or any other animal species;
2. Stimulation of skeletal development in livestock, domestic animals or any other animal species in order to achieve increased growth for commercial or any other purpose;
3. Treatment of neoplasia or hyperplasia of bone or cartilage, in humans, livestock, domestic animals or any other animal species;
4. Suppression of growth of skeletal components in livestock, domestic animals or any other animal species in order to achieve decreased growth for commercial or any other purposes; and
5. Alteration of the quality or quantity of cartilage and/or bone for any other purpose in any animal species including humans.
In a broader sense, the potential uses for the Sox-9 or SOX-9 gene or its protein product fall into two broad categories, viz. (1) the promotion of bone and/or cartilage differentiation and/or growth, and (2) the suppression of bone and/or cartilage differentiation and/or growth. As such the gene or its protein product (or any part or combination of parts of either), can be described as a therapeutic agent. Thus, the therapeutic agent may be Sox-9 or SOX-9 DNA or DNA fragments alone or in combination with any other molecule, Sox-9 or SOX-9 protein or protein fragments alone or in combination with any other molecule, antibodies to Sox-9 or SOX-9 alone or in combination with any other molecule, sense or anti-sense oligonucleotides corresponding to the sequence of Sox-9 or SOX-9 (alone or in combination with any other molecule). The method of administration of the therapeutic agent will differ depending on the intended use and on the species being treated (see Mulligan, 1993, Science, 260, 926-932; Morgan et al., 1993, Ann. Rev. Biochem., 62, 191-217). Such methods may include:
(i) Local application of the therapeutic agent by injection (Wolff et al., 1990, Science, 247, 1465-1468), surgical implantation, instillation or any other means. This method may be useful where effects are to be restricted to specific bones, cartilages or regions of bone or cartilage. This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the therapeutic agent so as to increase the effectiveness of that treatment. This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of the therapeutic agent.
(ii) General systematic delivery by injection of DNA, oligonucleotides (Calabretta et al., 1993, Cancer Treat. Rev., 19, 169-179), RNA or protein, alone or in combination with liposomes (Zhu et al., 1993, Science, 261, 209-212), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem., 13, 390-405) or any other mediator of delivery. This method may be advantageous for all intended uses (1-5 above) whether or not the effect is intended to be targeted to specific tissues or parts of the body, and regardless of whether the intended result is the stimulation or inhibition or suppression of Sox-9 or SOX-9 gene or protein activity. Where specific targeting is required, this might be achieved by linking the agent to a targeting molecule (the so-called xe2x80x9cmagic bulletxe2x80x9d approach employing for example, an antibody), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the therapeutic agent, or of cells responsive to the therapeutic agent.
(iii) Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al., 1987, Mol. Cell Biochem., 7, 2745-2752, or of cationic lipids and polyamines: Rose et al., 1991, BioTech., 10, 520-525), infection, injection, electroporation (Shigekawa et al., 1988, BioTech., 6, 742-751) or any other way so as to increase the expression or activity of Sox-9 or SOX-9 (gene or protein) in those cells. The modification may be mediated by plasmid, bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993, Science, 260, 926-932; Miller, 1992, Nature, 357, 455-460; Salmons et al., 1993, Hum. Gen Ther., 4, 129-141) or other vectors, or other agents of modification such as liposomes (Zhu et al., 1993, Science, 261, 209-212), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem., 13, 390-405), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al., 1991, Science, 254, 1507-1512 and by Dhawan et al., 1991, Science, 254, 1509-1512. Treated cells may be delivered in combination with any nutrient growth factor, matrix or other agent that will promote their survival in the treated subject.